J. Biochem. Biotechnol. DOI 10.1007/s13562-016-0364-8

REVIEW ARTICLE

Biosynthesis and therapeutic implications of iridoid glycosides from genus: the road ahead

1 1 2 Varun Kumar • Rajinder Singh Chauhan • Chanderdeep Tandon

Received: 3 September 2015 / Accepted: 2 May 2016 Ó Society for Plant Biochemistry and Biotechnology 2016

Abstract Picrorhiza genus is emerging as an important ISPD 4-Diphosphocytidyl-2C-methyl-D-erythritol paradigm for herbal drug formulations due to its versatile synthase iridoid glycosides exhibition and robustness in the treat- ISPE 4-Diphosphocytidyl-2C-methyl-D-erythritol ment of diverse infections including hepatic amoebiasis, kinase cancer, malaria, ulcerative colitis and cerebral ischemia MECPS 2-C-methyl-D-erythritol 2,4-cyclopyrophosphate reperfusion injury. Owing to the superiority of these synthase bioactivities, iridoid glycosides from Picrorhiza have HDS 1-Hydroxy-2-methyl 2-(E)-butenyl-4- become a hot research area over the years. A metabolic pyrophosphate synthase pathway for the formation of iridoid glycosides has been proposed. However, some enzymes and genes of this route are still unidentified and demand the enumeration of facilitating pathways contributing to the biosynthesis of Introduction iridoid glycosides. This review summarizes the current knowledge of all naturally occurring iridoid glycosides Over the years, common liver disorders such as chronic from Picrorhiza, their biosynthesis and pharmacological hepatitis and fatty liver face problems in treatment with capabilities which could provide the insight into metabolic colchicines, interferon and corticosteroids due to their regulation and the basis for the development of new drugs. reflective side-effect incidences (Luper 1998). Hence, have been explored to treat liver disorders. The iri- Keywords Anti-inflammatory Á Antioxidant Á Kutkin Á doid glycosides derived from Picrorhiza genus showed Picrorhiza Á Picrosides extensive promises against liver disorders and this article has reviewed discoveries on this class of natural products Abbreviations published over the last 25 years. This provides the platform DXPS 1-Deoxy-D-xylulose-5-phosphate synthase for the researchers to identify the research gaps in the DXPR 1-Deoxy-D-xylulose-5-phosphate biosynthesis of picrosides along with the knowledge of reductoisomerase therapeutic implications which could present good alter- native to the available ones in the development of new drugs in future. Some of the recent reviews, however, have also documented the pharmacological properties of iridoid & Chanderdeep Tandon glycosides from Picrorhiza species but they did not cata- [email protected] logue the pharmacological properties of individual iridoid glycosides (Bhattacharjee et al. 2013; Mondal et al. 2013; 1 Department of Biotechnology and Bioinformatics, Jaypee University of Information Technology, Waknaghat, Solan, Sah and Varshney 2013). Moreover, we have also stressed HP 173234, India at the biosynthetic point of iridoid glycosides to infer 2 Department of Biotechnology, Amity Institute of critical evaluation, which in turn suggests further types of Biotechnology, Amity University, Noida 201313, India investigations. 123 J. Plant Biochem. Biotechnol.

Picrorhiza genus belongs to family Among these iridoid glycosides, ‘Kutkin’ also known as (formerly known as Scrophulariaceae) and comprises of ‘Picroliv’ obtained from 3 to 4 years old roots and rhi- two species, Picrorhiza kurroa Royle ex Benth and Pi- zomes of Picrorhiza kurroa, was launched as herbal drug crorhiza scrophulariiflora Pennell, which grow in the formulation which contained 60 % of P-I and Kutkoside in Himalayan Mountains (Bhandari et al. 2008). P. kurroa, 1:1.5 ratio (Dwivedi et al. 1992; Verma et al. 2009). commonly known as Kutki, Karru and Indian gentian is Moreover, other drug formulations containing P-I and P-II distributed in the north-western at an elevation are: Katuki (1.29 % P-I and 1.16 % P-II); Arogya (1.01 % of 3000–5000 m (Kumar et al. 2015a) while P. scrophu- P-I and 0.55 % P-II); Livocare (4.17 % P-I and 3.25 % lariiflora, commonly known as Nepalese kutki, is found in P-II) and Livplus (0.07 % P-I and 0.01 % P-II) (Pandit the alpine zone of Sikkim, Nepal and China at an eleva- et al. 2013a). Mainly the roots and rhizomes tissues of tion of 3000–4000 m (Bantawa et al. 2011). The plants Picrorhiza species are used for the herbal drug formula- are perennial herbs and self-regenerating but their unreg- tions. In P. kurroa, shoots and roots accumulate P-I and ulated over-harvesting and lack of organized cultivation P-II, respectively whereas rhizomes accumulate both P-I has listed them as ‘endangered’ by ‘The International and P-II (Pandit et al. 2013a). Union for Conservation of Nature and Natural Resources So, keeping in view the diverse therapeutic potential of (IUCN)’ (Nayar and Sastri 1990; Bantawa et al. 2011). Picrorhiza genus and threat to its extinction; the need to Recently, a new species of Picrorhiza genus i.e. P. tung- completely dig out the biosynthetic route and therapeutic nathii has been identified which is distributed at an ele- activities of its active constituent’s demands attention. In this vation of 3400–4550 m in the Himalayan regions of review, we have summarized the iridoid glycosides from Himachal Pradesh and Uttarakhand state, India. The Picrorhiza genus, including their biosynthesis and pharma- plants are locally known by the same names as of cological activities which were the focal point to describe. P. kurroa i.e. kutki and karvi. The current conservation status is unknown as the exact population of this species is still to be examined due to its misidentification with Biosynthesis of iridoid glycosides P. kurroa (Pusalkar 2014). The use of Picrorhiza species in the Ayurvedic system Picrosides have attracted prime attention among all the of medicine is well known and is official in ‘‘The Indian iridoid glycosides present in Picrorhiza genus and thus, it Pharmacopoeia’’ and ‘‘Indian Herbal Pharmacopoeia’’ is necessary to understand how they are synthesized in Handa et al. (1998). The pharmaceutical activities are nature. Picrosides are monoterpenes (C10) attached with a attributed to iridoid glycosides, the active constituents of glucose molecule. The biosynthesis of picrosides proceeds these plants (Viljoen et al. 2012). So far, only 22 different via geranyl pyrophosphate (GPP), which is a monoterpene iridoid glycosides have been reported from Picrorhiza synthesized by the fusion of active isoprene units i.e. plants. Out of these, only 7 iridoid glycosides are present isopentenyl pyrophosphate (IPP) and its isomer dimethy- solely in P. kurroa viz. kutkin, kutkoside, picroside V (P- lallyl pyrophosphate (DMAPP), the two universal C5 V), boschnaloside, Bartsioside, mussaenosidic acid and building blocks. The production of these building blocks pikuroside (Basu et al. 1970; Jia et al. 1999; Mandal and can be achieved by two routes: the mevalonate pathway Mukhopadhyay 2004; Bhandari et al. 2010; Kumar et al. (MVA) and the methylerythritol phosphate (MEP) pathway 2013) as shown in Fig. 1, while 6 iridoid glycosides are (Laule et al. 2003). solely present in P. scrophulariiflora viz. amphicoside, The mevalonate pathway, first discovered in 1950s, piscroside A, piscroside B, verminoside, specioside and consists of six steps that start from acetyl CoA and pro- catalposide (Daqi et al. 1993; Li et al. 1998; Huang et al. ceeds through the intermediate mevalonic acid to produce 2006) (Fig. 2). On the other hand, 9 iridoid glycosides are IPP which is isomerised to DMAPP in the presence of an present both in P. kurroa and P. scrophulariiflora viz. IPP isomerase (Lange et al. 2000). On the other hand, an picroside I (P-I), picroside II (P-II), picroside III (P-III), alternative route to IPP i.e. MEP pathway was discovered picroside IV (P-IV), veronicoside, minecoside, 6-feruloyl- in 1990s (Rohmer et al. 1993), that starts from the con- catalpol, catalpol and aucubin (Stuppner and Wagner 1989; densation of glyceraldehyde-3-phosphate and pyruvate to Li et al. 1998; Mandal and Mukhopadhyay 2004; Huang produce DXP as the first intermediate. The key evidence et al. 2006; Bhandari et al. 2010; Kumar et al. 2013; Patil for the involvement of MVA and MEP pathways in et al. 2013; Sah and Varshney 2013) (Fig. 3). These iridoid biosynthesis of picrosides was provided by the gene glycosides demonstrate an assortment of bioactivities such expression analyses performed under differential condi- as antiinflammatory, antitumor, antioxidative, antiasthma, tions of picrosides accumulation in P. kurroa (Pandit et al. choleretic, immunostimulatory and hepatoprotective which 2013b). Moreover, the gene expression patterns vis-a`-vis are used to treat the liver disorders (Viljoen et al. 2012). P-I content were also observed in P. kurroa shoots at 123 J. Plant Biochem. Biotechnol.

Fig. 1 Chemical structure of iridoid glycosides present in P. kurroa different time intervals of plant growth at 15 °C under catalpol produced via Iridoid terpene biosynthetic route tissue culture conditions. The study revealed contribution and aromatic acids from shikimate/phenylpropanoid path- of MVA/MEP pathway genes (DXPS, ISPD, HMGR and way as shown in Fig. 4. The detection of intermediate PMK) for the first 20 days of plant growth with the major metabolites of catalpol biosynthetic pathway (boschnalo- role of MEP pathway as compared to the MVA pathway. side, bartsioside, mussaenosidic acid, aucubin and catalpol) Conversely, geraniol-10-hydroxylase (G10H) and DAHPS in P. kurroa using LC/ESI–MS/MS method indicated that contributed to P-I biosynthesis at 30 days of plant growth the picrosides biosynthesis proceeds via the proposed (Kumar et al. 2015b). Further, application of hydrogen biosynthetic route (Kumar et al. 2013). Nevertheless, peroxide and abscisic acid up-regulated the genes, PkDXS, enzymes involved in the picrosides biosynthesis are still PkHDR, PkCOMT and PkIPPI in congruence with picro- largely unresolved. The biosynthesis of picrosides initially sides accumulation in P. kurroa (Singh et al. 2013). These involves geraniol synthase (GES) which catalyses the analyses provide clues that up-regulation of above men- dephosphorylation of GPP to form geraniol. The geraniol is tioned genes might elevate the levels of GPP but they did then hydroxylated to form 10-hydroxygeraniol by G10H. not positively identify the activity of enzymes which are This NADPH-dependent cytochrome P450 has been being encoded by these genes. Functional validation of heterologously expressed in yeast and shown to catalyse above selected genes by either silencing or overexpression the first committed step of iridoid terpene biosynthesis in can make a strong contribution towards elucidating their Catharanthus roseus (Collu et al. 2001). A reversible functions in picrosides biosynthesis. dehydrogenation and subsequent oxidation of 10-hydrox- The picrosides biosynthesis beyond GPP has not been ygeraniol leads to formation of 10-oxogeranial by 10-hy- yet fully elucidated. Recently, a pathway for picrosides droxygeraniol oxidoreductase. The reduction of biosynthesis has been proposed in P. kurroa which repor- 10-oxogeranial in a NADH/NADPH dependent manner ted that all picrosides viz. P-I, P-II, P-III, P-IV, ver- with a subsequent cyclization step leads to formation of minoside and specioside are derived from esterification of iridodial by iridoid synthase (Geu-Flores et al. 2012). The

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Fig. 2 Chemical structure of iridoid glycosides present in P. scrophulariiflora next enzyme, which is responsible for constructing the various substrates helps in the prediction of preferred iridotrial and 7-deoxyloganetic acid from an iridodial substrate for the enzyme, albeit does not guarantee that the intermediate, is an iridoid oxidase. Miettinen et al. (2014) substrate has been correctly identified. Thus, a meticulous functionally characterized iridoid oxidase from C. roseus analysis of the above enzyme which appears to be an UDP- and reconstituted it in Nicotiana benthamiana which glucosyltransferase is needed as it is still not well charac- revealed its essentiality for iridoid biosynthetic pathway. A terized at the enzymatic level. An enzymatic reaction can glucosylation step follows the formation of iridotrial and be considered to be ‘proven’ only when the enzyme 7-deoxyloganetic acid which is catalysed by UDP-gluco- responsible for the catalysis of the given step catalyses the syltransferase. Bhat et al. (2013) identified an UDP-glu- desired reaction (Fridman and Pichersky 2005). cosyltransferase (UGT94F2) which showed positive The next steps of picrosides biosynthesis involve relationship with picroside content in different tissues of hydroxylation, dehydration, decarboxylation, epoxidation P. kurroa upon elicitor treatment and seemed to be and esterification reactions (Fig. 4). These steps are still involved in the conversion of 7-deoxyloganetin to epi- not characterized at enzymatic level. On the basis of bio- deoxyloganic acid. This investigation adds complication in chemical reactions, it could be hypothesised that cyto- picrosides biosynthesis by providing an alternative route to chrome p450s along with decarboxylase, dehydratase and the biosynthesis of picrosides which involves 7-deoxylo- acyltransferase might catalyse the desired steps. The ganetin instead of boschnaloside as an intermediate in the tedious purification of cytochrome p450s from crude plant formation of epi-deoxyloganic acid. The correct identity of extracts to homogeneity followed by protein sequencing the step involved or the possibility of involvement of both requires standardized enzymatic assay which would be routes in the biosynthesis of picrosides must be fed with hampered by very low levels of secondary metabolic more evidences. However, docking of enzymes with enzymes which are even five to six order lower in

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Fig. 3 Chemical structure of iridoid glycosides present in both P. kurroa and P. scrophulariiflora magnitude as compared to their respective substrates in This approach seems to be more efficient in the identi- planta (Weitzel and Simonsen 2015). Further, accessibility fication of missing enzymes in biosynthetic pathways but to the intermediates of picrosides biosynthetic pathway is must be ensured with in depth biochemical characterization limited. Some of the intermediate metabolites are available of all the isolated enzymes. but others require chemical synthesis which can be costly Considerable efforts have also been focused on the and laborious. identification of proteins involved in picrosides biosyn- To circumvent this problem, current research efforts for thesis in P. kurroa by using differential proteomics study the identification of missing enzymes have been focused on under metabolite accumulating and non-accumulating the use of transcriptomic data to isolate full length cDNAs conditions. Sud et al. (2013, 2014) identified eight differ- by random approach and heterologously expressed in entially expressed proteins in P. kurroa shoot samples Escherichia coli or yeast to reveal their substrate speci- while revealed 21 differentially expressed proteins from ficity. Bhat et al. (2014) heterologously expressed a stolon and roots of P. kurroa using MALDI-TOF MS fol- NADPH-cytochrome P450 reductase in E. coli, which was lowed by MASCOT database search. This subject still isolated from P. kurroa and dedicated this enzyme to needs further attention and could be supplemented with picroside biosynthesis due to its positive correlation with temporal patterns of proteins expression vis-a`-vis target picroside content in samples collected at varied altitudes. metabolite accumulation in order to give a strong indica- Cytochrome P450 reductases are known to serve as elec- tion of involvement of these genes in picrosides biosyn- tron donor to cytochrome P450s which are supposed to be thesis. Additionally, the researchers would also endeavour major enzymes that catalyze the picroside biosynthesis in different subcellular organelle extracts in order to identify P. kurroa. the different cytochrome P450s in P. kurroa. Owing to the

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123 J. Plant Biochem. Biotechnol. b Fig. 4 Metabolic pathway showing biosynthesis of iridoid glycosides antioxidant properties which increased the rate of gastric in P. kurroa. The metabolic network was reconstructed including ulcer healing in rats (Ray et al. 2002). The extract mevalonate/non-mevalonate, shikimate/phenylpropanoid and iridoid biosynthetic pathways. Solid and dotted arrows indicate the single enhanced in vivo free radical scavenging action by and multiple steps, respectively. Question marks indicate enzymatic restoring the superoxide dismutase and catalase enzyme’s steps with no available information activities along with inhibition of lipid peroxidation. A detailed analysis of mode of action revealed that healing of limited database of plant proteins along with the low gastric ulcer in rats was also associated with increase in

sequence similarity of cytochrome P450s, searching of mucus production, prostaglandin E2 levels, expressions of strictly conserved regions for cytochrome P450s in the cyclooxygenase isoforms (COX-1 and COX-2), and growth hypothetical proteins i.e. the four-helix form (D, E, I and promoting factors viz. vascular endothelial growth factor L), could provide further elucidation of cytochrome P450s (VEGF) and epidermal growth factor, EGF (Banerjee et al. in P. kurroa (Weitzel and Simonsen 2015). Further, inte- 2008). Further, the root extract of P. kurroa at a dose of gration of proteomics with transcriptomics could lead to a 200 mg/kg body weight, exhibited cardioprotective effect rapid elucidation of cytochrome P450s involved in the by restoring myocardial superoxide dismutase, catalase, biosynthesis of picrosides but it must be clear that only and glutathione peroxidase enzymes which resulted in the enzymatic activity can unambiguously assign the function attenuation of isoproterenol-induced oxidative stress to protein. Interestingly, the effect of primary metabolism (Nandave et al. 2013). Continuing investigation on on the accumulation of secondary metabolites was antioxidant activity of P. kurroa extracts revealed that observed by correlating the catalytic activities of the rate butanol and ethyl acetate extracts were more efficient in limiting enzymes of primary metabolic pathways viz. scavenging of free radicals as compared to ethanol extract hexokinase, pyruvate kinase, isocitrate dehydrogenase, (Kant et al. 2013). On the other hand, Rajkumar et al. malate dehydrogenase and NADP-malic enzyme with their (2011) observed that both methanolic and aqueous extracts gene expression under differential conditions of picrosides of P. kurroa rhizome exhibited antioxidant potential by accumulation in P. kurroa (Kumar et al. 2016). This pro- scavenging 2,2-diphenyl-1-picrylhydrazyl (DPPH) and vides broader targets for metabolic engineering in order to inhibiting lipid peroxidation. attain enhanced secondary metabolite production. Picrorhiza kurroa extract was also evaluated for anti- In summary, the enzymes for six of the fifteen steps carcinogenic activity which showed significant inhibition involved in the biosynthesis of picrosides in P. kurroa, of N-nitrosodiethylamine (NDEA) induced hepatocarcino- have been identified. Despite this impressive progress in genesis by reducing the levels of glutathione S-transferase the elucidation of biosynthetic pathway for picrosides in (GST), aniline hydroxylase (AH) and gamma-glutamyl P. kurroa, a lot of work is still required to identify the transpeptidase (GGT) in a dose dependent manner (Jeena missing enzymes along with the key enzymes involved in et al. 1999). Similarly, administration of P. kurroa extract picrosides biosynthesis in order to control the flux of significantly inhibited sarcoma induced by 20-methyl- intermediates through the pathway that will eventually cholanthrene (20 MC) in mice along with a significant benefit the biotechnological production of picrosides. increase in the life span of mice bearing transplanted tumour and thus exhibited both anti-tumour and anticar- cinogenic activities (Joy et al. 2000). Pharmacological activities of iridoid glycosides The antimalarial potential of P. kurroa extracts was observed by significant inhibition of Plasmodium bergheii In this section, we have presented an exhaustive compila- (Singh and Banyal 2011). Further, the examination of tion of the pharmacological properties of Picrorhiza extract P. scrophulariiflora extract along with its ten isolated pure along with its all iridoid glycosides in order to look into compounds, showed that 10 mg/mL concentration of crude their therapeutic potential (Table 1). extract inhibited 95 % P. falciparum 3D7 malaria parasites and the compound 10 solely accounted for the antimalarial

activity (IC95 value of 23.5 lM) (Wang et al. 2013a, b). Picrorhiza plant extracts The investigations on antimicrobial potential of Pi- crorhiza revealed that methanolic extract of P. kurroa Picrorhiza genus has been used in the treatment of various rhizomes exhibited a significant activity against E. coli, disorders as recorded by a wide range of bioactivities. The B. subtilis and S. aureus, bacterial strains when compared dried rhizome of P. scrophulariiflora was traditionally used with a standard drug Ciprofloxacin (Sharma and Kumar to remove damp-heat and to relieve consumptive fever 2012). (Pharmacoepia 1995). Further, the extract of P. kurroa The immunostimulatory activity was observed in rhizome at a dose of 20 mg/kg body weight exhibited experimental animals upon treatment with 50 % ethanolic 123 J. Plant Biochem. Biotechnol.

Table 1 Pharmacological properties of iridoid glycosides from Picrorhiza S. Bioactivities Conc. of active compound Model Iridoid Reference No. used glycoside

1 Anti-leishmanial 10 mg/kg for 12 days, oral In vivo Kutkin Sane et al. 2011 2 Neuritogenic 60 lM In vitro P-I; P-II Li et al. 2000; 2002 3 Anticancer 5 lM and 10 lM In vitro P-I; kutkoside; Rathee et al. 2013 kutkin 4 Immunostimulant 10 mg/kg for 7 days, oral In vivo Kutkin Puri et al. 1992 5 Neuroprotective 10 mg/kg, 250 ll intravenously at In vivo P-II Li et al. 2010; Guo et al. 2010 the end of ischemic 2 h before reperfusion 22 h 20 mg/kg, intraperitoneally at In vivo Pei et al. 2012 ischemia 1.5 h 10–20 mg/kg, intraperitoneally at In vivo Zhao et al. 2014; 2013; Yang ischemia 1.5–2 h et al. 2014 6 Hepatoprotective 5, 10, 20 mg/kg for 7 days, In vivo P-II Gao and Zhou 2005a, b intragastrically 0.005, 0.01, and 0.02 mmol/L In vitro 12 mg/kg for 7 days, oral In vivo Kutkin Dwivedi et al. 1992 6 and 12 mg/kg for 7 or 8 days, In vivo Ansari et al. 1991 oral 12 mg/kg/day for 15 days, oral In vivo Rastogi et al. 1996 25 mg/kg/day for 15 days, oral In vivo Rastogi et al. 2000 12 mg/kg for 7 days, oral In vivo P-I Dwivedi et al. 1992 12 mg/kg for 7 days, oral In vivo Kutkoside Dwivedi et al. 1992 7 Protection against hypoxia/ 50–200 lg/ml for 48 h In vitro P-II Meng et al. 2012a;b reoxygenation injuries 0–50 lg/ml for 24 h In vitro Kutkin Gaddipati et al. 1999 8 Hepatic amoebiasis 12 mg/kg for 7 days, oral In vivo Kutkin Singh et al. 2005 9 Prevention of myocardial ischemia 1, 10 and 100 lM In vitro P-II Wu et al. 2014 reperfusion injury 10 Hypolipidaemic 25 mg/kg for 30 days, oral In vivo Kutkin Khanna et al. 1994 11 Anti-allergic and anti-anaphylactic 6.25–25 mg/kg for 4 days, oral In vivo Kutkin Baruah et al. 1998 12 Ulcerative colitis 12.5 mg/kg for 7 days, oral In vivo Kutkin Zhang et al. 2012

extract of P. kurroa leaves due to the stimulation of Picroside II phagocytosis along with cell-mediated and humoral com- ponents of the immune system (Sharma et al. 1994). In P-II showed neuroprotective properties by reducing the addition, experimental investigations showed that P. kur- expressions of Caspase-3 and PARP and thus inhibited the roa significantly reduced the lipid content, glutamic neuronal apoptosis induced by cerebral ischemia reperfu- oxaloacetate transaminase (GOT) and glutamic pyruvate sion injury (Li et al. 2010). Further, it was observed that transaminase (GPT) in liver injury induced by galac- inhibition of neuronal apoptosis was the result of antioxi- tosamine in rats. This was also supported by clinical trials dant and anti-inflammatory role of P-II. The anti-inflam- which showed the efficiency of P. kurroa in viral hepatitis matory mechanism of P-II showed reduced expressions of hepatoprotection in animal model (Vaidya et al. 1996). Toll-like receptor 4 (TLR4), nuclear factor jB (NFjB) and Further findings reported that administration of hydroal- tumor necrosis factor a (TNFa) (Guo et al. 2010). More- coholic extract of P. kurroa for a period of 4 weeks sig- over, the optimization of therapeutic dose and time window nificantly lowered the lipid content (mg/g) of liver and of P-II revealed that 20 mg/kg body weight P-II should be thus, has the potential for treatment of non-alcoholic fatty injected intraperitoneally at ischemia 1.5 h in treating liver disease (Shetty et al. 2010). Moreover, the ethanolic cerebral ischemic injury (Pei et al. 2012). Further treatment extract of P. scrophulariiflora prevents diabetic with 10–20 mg/kg body weight P-II at ischemia 1.5 h nephropathy via inhibition of redox-sensitive inflammation recorded the reduced levels of free radicals in cerebral (He et al. 2009). ischemic injury along with the reduction in malonaldehyde 123 J. Plant Biochem. Biotechnol. content and an improvement in superoxide dismutase expression similar to the previous finding of Meng et al. activity (Zhang et al. 2013). This suggests that antioxidant (2012b) in order to mediate anti-apoptotic effect. potential of P-II could reduce the oxidative damage induced by cerebral ischemic injury. Further, treatment with P-II significantly decreased the concentrations of Kutkin/picroliv malondialdehyde, nitric oxide and hydrogen peroxide in brain tissue along with an increase in the activities of Picroliv/Kutkin showed promising hepatoprotective activ- superoxide dismutase, glutathione peroxidase (GSHPx) and ity against hepatotoxic effects induced by paracetamol and catalase (Yang et al. 2014). This implied the neuroprotec- galactosamine in adult male albino rats (Ansari et al. 1991). tive role of P-II with the optimal therapeutic dose and time The administration of 12 mg/kg/day picroliv for 7 days window as injecting 10–20 mg/kg intraperitoneally at protected liver by preventing the biochemical changes in 1.5–2.0 h. Further, the neuroprotective effect of P-II was liver and serum of galactosamine-toxicated rats (Dwivedi also observed due to increased mRNA and protein et al. 1992). Moreover, administration of picroliv along expression of myelin basic protein (MBP) and arrangement with alcohol for 15 days subsequent to an initial alcohol of myelin fibres in order with optimized therapeutic dose exposure for 30 days resulted in reduced biochemical and time window (Zhao et al. 2014). Moreover, the opti- parameters in liver and serum of albino rats (Rastogi et al. mized therapeutic dose of 10–20 mg/kg body weight P-II 1996). Further, picroliv altered biochemical parameters in a at ischemia 1.5–2.0 h was observed for anti-inflammatory dose-dependent manner (36–100 %). Its anticholestatic effect (Zhao et al. 2013). property results in the prevention of bile flow, reduction in P-II was also observed to relieve hepatocyte injuries bile salts and bile acids. It also increases the activity of induced by carbon tetrachloride, D-galactosamine and aldehyde dehydrogenase and acetaldehyde dehydrogenase acetaminophen in mice. Hepatic injury was associated with which impairs with acetaldehyde metabolism whose high levels of alanine aminotransferase and aspartate accumulation was mainly responsible for hepatic injury aminotransferase which were decreased upon administra- (Saraswat et al. 1999). Further, the hepatoprotective effect tion of P-II. In addition, P-II scavenged free radicals and of picroliv was revealed against rat hepatotoxicity induced modulated the balance of liver energy metabolism by by aflatoxin B1. Picroliv administration resulted in the increasing ATPase activity in mitochondria and thereby reversal of increase in tau-glutamyl transpeptidase, 50-nu- producing hepatoprotective effects (Gao and Zhou 2005a). cleotidase, acid phosphatase and acid ribonuclease activi- Further investigations revealed the role of bcl-2 and bax ties induced in liver and serum of rats treated with aflatoxin genes in hepatocytes protection against apoptosis. The B1 (Rastogi et al. 2000). Further, Singh et al. (2005) study showed up-regulation of bcl-2 and bcl-2/bax ratio on revealed prevention of Entamoeba histolytica induced administration of 10 mg/kg P-II in a dose dependent hepatic damage by picroliv. It provides protection in the manner (Gao and Zhou 2005b). elevated levels of glutamic oxaloacetate transaminase Liu et al. (2007) observed that P-II along with nerve (60.7 %), glutamic pyruvate transaminase (87 %) and growth factor (NGF) (2 ng/mL) significantly reduced alkaline phosphatase (61.37 %); induced by treatment of reactive oxygen species (ROS) levels, leading to the syn- carbon tetrachloride followed by E. histolytica infection. ergistic protective effect in vitro on PC12 cells against Similarly, the reversal of changes in glutamic oxaloacetate hydrogen peroxide induced oxidative stress. Furthermore, transaminase, glutamic pyruvate transaminase and alkaline P-II induced reduction of ROS levels along with enhanced phosphatase enzyme activities upon administration of activities of antioxidant enzymes (SOD and GSH-Px) and picroliv in dose dependent manner was reported against calcium level demonstrated its protective effect against thioacetamide-induced hepatic damage in the rats (Visen cardiomyocyte injury induced by hypoxia/reoxygenation et al. 1991). Further, the efficiency of picroliv has been (Meng et al. 2012a). The P-II treatment increased the shown to be comparable with silymarin for preventing expression of bcl-2 but decreased the expression of bax galactosamine, paracetamol, thioacetamide and carbon along with the activation of PI3K/Akt and cAMP response tetrachloride induced hepatic damage (Verma et al. 2009). element-binding protein (CREB) pathways which inhibited The potential of picroliv has also been observed for caspase-3 activation and finally attenuated myocyte apop- scavenging oxygen free radicals. Picroliv in addition to P-I tosis (Meng et al. 2012b). Interestingly, P-II treatment and kutkoside, inhibited the xanthine oxidase activity prevents myocardial ischemia reperfusion injury (MIRI) in thereby preventing the generation of superoxide anions rats (Wu et al. 2014). It increases the production of nitric along with malondialdehyde (MDA) generation in rat liver oxide mediated by activation of PI3K/Akt/eNOS signalling microsomes (Chander et al. 1992). An investigation of the pathway along with the modulation of bcl-2 and bax genes picroliv potential as immunostimulant revealed that 10 mg/

123 J. Plant Biochem. Biotechnol. kg picroliv administration for 7 days showed significant activated protein (MAP) kinase (Li et al. 2000). Further, elevation in macrophage migration index, [14C]-glu- enhanced neurite outgrowth from PC12D cells was also cosamine uptake, chemiluminescence of peritoneal mac- induced by basic fibroblast growth factor (bFGF), stau- rophages and higher uptake of [3H]-thymidine in the rosporine and dibutyryl cyclic AMP (dbcAMP) through lymphocytes of treated mice along with providing protec- activation of MAP kinase-dependent signaling pathway tion against Leishmania donovani infection in golden upon P-I and P-II treatment (Li et al. 2002). hamsters (Puri et al. 1992). An enhancement in the inhi- bition of L. donovani parasite was observed when picroliv was administered in combination with antileishmanial Catalpol drugs viz. paromomycin and miltefosine, which signifi- cantly increased the phagocytosis and lymphocyte prolif- Catalpol showed anti-leishmanial as well as topoisomerase eration responses (Sane et al. 2011). inhibiting activities (Mandal and Mukhopadhyay 2004). Picroliv also reduces the serum lipids levels in hyper- The pharmacological action of catalpol has not been fully lipaemia induced by triton WR-1339 and cholesterol. This established, but it might be involved in stimulation of hypolipidaemic action of picroliv was mediated through secondary metabolites production (Kumar et al. 2013). cholesterol biosynthesis inhibition along with increase in excretion of faecal bile acid and cholesterol acyltransferase activity (Khanna et al. 1994). Further, picroliv was Conclusions and future directions observed to be associated with augmentation of human T cell response in mycobacterial diseases (Sinha et al. 1998). The iridoid glycosides derived from Picrorhiza genus At a dose of 25 mg/kg p.o., picroliv caused 82 % inhibition harbour a wide spectrum of high-value pharmacological of passive cutaneous anaphylaxis in mice and 50–85 % in potential including antioxidant, anti-inflammatory, anti- rats along with the protection of mast cells from degranu- malarial, antimicrobial, anticancer, anticholestatic and lation thereby exhibited anti-allergic and anti-anaphylactic hepatoprotective activities. With so much bioactivity, a properties (Baruah et al. 1998). Picroliv acts as protective meticulous analysis of iridoid glycosides biosynthesis has agent against injuries induced by hypoxia/reoxygenation been observed in Picrorhiza genus. The incorporation by reducing the lactate dehydrogenase release in Hep 3B studies over the years demonstrated the involvement of and Glioma cells. The picroliv treatment inhibited protein MEP, MVA, phenylpropanoid and monoterpenoid path- tyrosine kinase activity which led to tyrosine dephospho- ways in biosynthesis of picrosides but the sporadic rylation of several proteins along with the reduction in knowledge of enzymes in the their biosynthesis provenance protein kinase C (PKC), indicating the involvement of a distinct challenges in pathway analysis. Nevertheless, plant novel signal transduction pathway in hypoxia/reoxygena- tissue culture has been standardised for picrosides pro- tion induced injuries (Gaddipati et al. 1999). duction; however, P-I content is much lower (*5-fold) and The antioxidant and anti-inflammatory properties of P-II is not detected in tissue cultured plants as compared to picroliv play a critical role in ulcerative colitis. The plants grown in natural habitats. Thus, metabolic engi- administration of picroliv caused subsequent amelioration neering may be exploited to maximize picrosides content in in disease activity index along with the reduction in cellular systems by redirecting the carbon flux towards enhanced myeloperoxidase activity, MDA concentration, picrosides biosynthesis. Consequently, it is hard to decide and the IL-1b, TNF-a, and NF-jB p65 expressions in the strategy for enhancing the P-I content in P. kurroa dextran sulfate sodium induced ulcerative colitis in mice through metabolic engineering. Hence, the incorporation of (Zhang et al. 2012). Further investigations documented the remaining enzymes of picroside biosynthetic pathway anticancer potential of picroliv which along with P-I and along with additional constraints such as identification of kutkoside from P. kurroa down-regulated MCF-7 cell lines key steps, metabolic control analysis and thermodynamics invasion and migration via reduced matrix metallopro- would expand the scope of novel drugs development. This teinases (MMPs) expressions (Rathee et al. 2013). will result in the identification of rate limiting enzymes along with the knowledge of mechanisms and regulation of pathway which will benefit the biotechnological production Picroside-I of picrosides.

P-I along with P-II exhibited neuritogenic activity at a Acknowledgments Financial support of this work was provided by concentration of 60 lM in the presence of 2 ng/mL, NGF. Department of Biotechnology (DBT), Ministry of Science and Technology, Govt. of India in the form of a programme support on This significantly enhanced neurite outgrowth induced by high value medicinal plants under Centre of Excellence. NGF from PC12D cells through amplifying a mitogen- 123 J. Plant Biochem. Biotechnol.

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